Title: Development of an improved core technology for efficient genetic code expansion in biomedical research Principal Investigator: Ryan Mehl Summary Genetic code expansion (GCE) allows the introduction of noncanonical amino acids (ncAAs) into proteins. This provides a powerful toolbox to manipulate and expand biochemical activities, opens new possibilities to control protein function in cells, and creates new therapeutics against many human pathologies, including cancer, autoimmune syndromes, and metabolic diseases. First developed in bacterial systems, biomedical applications of GCE are now rapidly increasing as a consequence of recently expanded abilities in both mammalian cell culture and model multicellular organisms. Despite these advances and the enormous potential of the technology, a key challenge to fully implementing GCE ? especially for in vivo applications ? is the inefficient synthesis of ncAA-containing proteins in recoded bacterial and mammalian cells. In particular, a variety of studies in recent years have converged on identifying the key bottleneck in the technology: creating efficient pairs of orthogonal aminoacyl-tRNA synthetases (o-RS) and transfer RNAs (o-tRNAs). Indeed, studies indicate that existing o-RS/tRNA pairs are two to five orders of magnitude less efficient than naturally occurring pairs. Our goal in this proposal is to eliminate this bottleneck by (1) developing a generally applicable approach for optimizing the function of engineered o-RS/tRNA pairs, and (2) creating improved ?high-efficiency? chassis for current workhorse o-RS/tRNA pairs. With these advances, in vivo GCE studies to probe cellular functions can be carried out in ways that generate proteins containing one or more ncAAs at similar concentrations and regulatory control as in natural cells, and do it consistently without massive amounts of truncation products or excess GCE components that cause confounding disturbances. No one has yet attempted to systematically attack this problem. Our approach has two key features as compared to all prior work in the field. First, we will generate a novel, detailed database correlating in vitro kinetic properties of purified o-RS/tRNA with the performance of the same o-RS/tRNA pairs in protein synthesis in vivo and in cell-free bacterial and mammalian extracts. Second, we will develop and apply an iterative process to improve existing o- RS/tRNA pairs, guided by both computational approaches and experimental determination of allosteric pathways, in order to optimize the design of libraries covering portions of the protein-tRNA complexes outside the well-explored primary ncAA binding site. These libraries are then incorporated into directed evolution approaches to select for catalytically improved pairs. We propose this innovative approach in response to a specific NIGMS funding opportunity focused on technology development to enable biomedical research, and it fully embodies the qualities specified by PAR-17-045 for the creation of broadly applicable new research tools that are higher-risk ventures with truly transformational potential.